Knowledge of the number of molecules of a particular antigen on a cell has been of increasing interest. This value is most often approximated on flow cytometry by measurement of the number of antibody molecules bound to that antigen on the cell. Such measurement requires that the flow cytometer to be calibrated.
Various approaches have been used to convert flow cytometric data of cell-bound antibody to the number of copies of antibody per cell. These include: radioactive antibody plus fluorescent second step; comparison to commercial calibration beads; and by analysis of binding curves from fluorescent antibody titrations. These different approaches have yielded variable copy numbers for antibodies bound to lymphocyte antigens. For example, reports of CD4 antibody vary from 46,000 to 132,000 copies per cell, CD8 antibody from 130,000 to 430,000 copies per cell, and CD45 antibody from 180,000 to 970,000 copies per cell. Further work on the basic methodology for broadly applicable standards was thus warranted.
A broadly applicable and intrinsically accurate standard would appear to be R-PE beads. Other approaches have certain drawbacks: 1) fluorescein is some 10-fold less bright than R-PE, self-quenches when molecules are brought into close proximity to each other, and therefore the number of fluoresceins conjugated per antibody molecule does not necessarily reflect fluorescence intensity; 2) the use of calibration beads with a known number of anti-mouse Ig binding sites is problematic due to different efficiencies of binding of different monoclonals, resulting in copy number determinations that vary by as much as 2-fold; 3) the use of radioactive antibody plus polyclonal fluorescein-labeled second step is problematic because the stoichiometry of second step to monoclonal varies with different monoclonals and may vary from lot to lot and depending upon the source of the polyclonal second step. In contrast, R-PE gives high sensitivity for low copy number antigens, R-PE beads alleviate the uncertainty in binding efficiency of antibody conjugates to anti-mouse beads, and R-PE conjugates are available as 1:1 conjugates thus giving a more straightforward relationship between R-PE copy number and antigen copy number. However, determining the amount of R-PE on a bead by direct fluorescence measurement suffers from the drawback that the presence of particles in suspension can interfere with that measurement.